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Method and nucleic acids for the analysis of a lung cell proliferative disorderRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidMethod and nucleic acids for the analysis of a lung cell proliferative disorder description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128592, Method and nucleic acids for the analysis of a lung cell proliferative disorder. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The levels of observation that have been studied by the methodological developments of recent years in molecular biology, are the genes themselves, the translation of these genes into RNA, and the resulting proteins. The question of which gene is switched on at which point in the course of the development of an individual, and how the activation and inhibition of specific genes in specific cells and tissues are controlled is correlatable to the degree and character of the methylation of the genes or of the genome. In this respect, pathogenic conditions may manifest themselves in a changed methylation pattern of individual genes or of the genome. [0002] The present invention relates to nucleic acids, oligonucleotides, PNA-oligomers, and to a method for the analysis of lung cell proliferative disorders, the differentiation between subclasses of said disorder or the detection of a predisposition to said disorders, by analysis of the genetic and/or epigenetic parameters of genomic DNA and, in particular, with the cytosine methylation status thereof. [0003] Lung cancer is among the most commonly occurring malignancies in the world and is one of the few that continues to show an increasing incidence. In men it is the leading cause of of death in Western countries. In 2000, the incidence in the US is estimated to be 164 000 new cases and 157 000 deaths from the disease. 5 year survival rates are only 14% in the US (Ginsberg et al., Principles & Practice of Oncology. 6.sup.th Edition). The most prominent risk factor is smoking, around 80% of lung cancer deaths among men and 75% among women are likely to be attributable to smoking (Minna et al., Cancer: principles and practice of oncology, 3.sup.rd ed., 1989). [0004] Lung cancer falls into two major histologic classes, small cell lung cancer and non-small cell lung cancer. The latter one represents 82% of lung cancer cases (Murren et al., Principles & Practice of Oncology. 6.sup.th Edition) and can be further subclassified into squamous cell carcinoma, once the most frequent of all lung cancers in North America, and adenocarcinoma, to which 40% of new lung cancer cases can be attributed (Ginsberg et al., Principles & Practice of Oncology. 6.sup.th Edition). Squamous cell carcinoma arises most frequently in the proximal segmental bronchi. Because of the ability of squamous cells to exfoliate, this tumour can be detected by cytologic examination of sputum. Adenocarcinoma usually arises more peripherally and has a somewhat worse prognosis compared to squamous cell carcinoma. [0005] Because of the poor prognosis of lung cancer, identification of patients at an early stage, where the disease can still be cured, is of outstanding importance. Currently, most patients present with metastatic (stage IV) disease (Ginsberg et al., Principles & Practice of Oncology. 6.sup.th Edition). Sputum or bronchoalveolar lavage analysis, imaging techniques from conventional chest radiography to spiral computed tomography, percutaneous fine-needle aspiration, bronchoscopy are used to diagnose patients in whom the disease is suspected. Whereas helical computed tomographic scans are particularly successful in picking up small peripheral adenocarcinomas that cannot yet be visualised by standard chest x-rays, cytologic examination of sputum provides a high sensitivity for central squamous cell lesions. However, because of their invasiveness, radiation exposure and, above all, the high number of false positives, these methods are currently only applied in a very small subset of individuals known to be at high risk for the disease or if symptoms are already present. [0006] In the last decade, knowledge has accumulated on molecular alterations which occur during progression from dysplasia or atypia to cancerous lesions of the lung. These alterations include chromosomal abnormalities such as deletions of 3p, 9p and 17p (Sekido et al., Principles & Practice of Oncology. 6.sup.th Edition), microsatellite instability (Sekido et al., Biochim Biophys Acta 1998, 1378: F21), activation of protooncogenes, e.g. EGFR, ERBB2, KIT, and MET (Rusch et al., Clin Cancer Res 1997, 3:515, Tsai et al., Cancer Res 1996, 56:206, Krystal et al., Cancer Res 1998, 58:4660), inactivation of tumor suppressor genes like p53 (Bennett et al., J Pathol 1999, 187:8), p16 (Sekido et al., Biochim Biophys Acta 1998, 1378: F21, Belinsky et al., PNAS USA 1998, 95: 11891) and RB (Reissmann et al., Oncogene 1993, 8:1913). One of the earliest molecular alterations in tumorigenesis is aberrant DNA methylation. In a recent study, Dai and coworkers were able to show that out of 1184 CpG islands screened by RLGS analysis up to 5.3% are methylated in some non-small cell lung cancers. In addition, aberrant methylation could be detected not only in the tumour itself, but also in different body fluids, such as serum (Esteller et al., Cancer Res, 1999, 59:67) and bronchoalveolar lavage samples (Ahrendt et al., J Natl Cancer Inst 91:332). [0007] Molecular markers offer the advantage that even samples of very small sizes and samples whose tissue architecture has not been maintained, e.g. very small biopsies or single cells can be analysed quite efficiently. In addition, molecular alterations identified in different tumour types can be detected also in body fluids such as serum, plasma, sputum or bronchoalveolar lavage, probably much earlier than cytological analysis. Detailed knowledge of the molecular pathogenesis of a disease also offers the possibility to develop new drugs targeted specifically at alterations occurring at a specific stage in the disease. [0008] Aberrant DNA methylation within CpG islands is common in human malignancies leading to abrogation or overexpression of a broad spectrum of genes (Jones, P. A. Cancer Res 65:2463-2467, 1996). Abnormal methylation has also been shown to occur in CpG rich regulatory elements in intronic and coding parts of genes for certain tumours (Chan, M. F., et al., Curr Top Microbiol Immunol 249:75-86,2000). Highly characteristic DNA methylation patterns could also be shown for breast cancer cell lines (Huang, T. H.-M., et al., Hum Mol Genet 8:459-470, 1999). Large-scale methylation analysis has not been applied to lymphomas so far, but alterations of the methylation of single genes have been described in several subtypes of Non-Hodgkin lymphoma, e.g. TCL1 (Yuille et al., Genes Chromosomes Cancer 2001, 30:336-41), p15 and AR (Baur et al., Blood. 1999, 94:1773-81, Martinez-Delgado et al., Leukemia 1998 12:937-41), the androgen receptor (McDonald et al., Genes Chromosomes Cancer. 2000 28:246-57), and the MyoD1 gene (Taylor et al., Leukemia. 2001, 15:583-9). [0009] 5-methylcytosine is the most frequent covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behaviour as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification. [0010] A relatively new and currently the most frequently used method for analysing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behaviour. However, 5-methylcytosine remains unmodified under these conditions. [0011] Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, can now be detected as the only remaining cytosine using "normal" molecular biological techniques, for example, by amplification and hybridisation or sequencing. All of these techniques are based on base pairing which can now be fully exploited. In terms of sensitivity, the prior art is defined by a method which encloses the DNA to be analysed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Using this method, it is possible to analyse individual cells, which illustrates the potential of the method. However, currently only individual regions of a length of up to approximately 3000 base pairs are analyzed, a global analysis of cells for thousands of possible methylation events is not possible. However, this method cannot reliably analyse very small fragments from small sample quantities either. These are lost through the matrix in spite of the diffusion protection. [0012] An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255. [0013] To date, barring few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 March-April; 5(2):94-8) the bisulfite technique is only used in research. Always, however, short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 November; 17(3):275-6) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L, Jones P A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO 95/00669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15; 25(12):2532-4). In addition, detection by hybridisation has also been described (Olek et al., WO 99/28498). [0014] Further publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 June; 16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb. 25; 22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5' region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97/46705, WO 95/15373, and WO 97/45560. [0015] An overview of the Prior Art in oligomer array manufacturing can be gathered from a special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), published in January 1999, and from the literature cited therein. [0016] Fluorescently labelled probes are often used for the scanning of immobilised DNA arrays. The simple attachment of Cy3 and Cy5 dyes to the 5'-OH of the specific probe are particularly suitable for fluorescence labels. The detection of the fluorescence of the hybridised probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available. [0017] Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-TOF) is a very efficient development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionisation of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct. 15; 60(20):2299-301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapour phase in an unfragmented manner. The analyte is ionised by collisions with matrix molecules. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones. [0018] MALDI-TOF spectrometry is excellently suited to the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57). The sensitivity to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size. For nucleic acids having a multiply negatively charged backbone, the ionisation process via the matrix is considerably less efficient. In MALDI-TOF spectrometry, the selection of the matrix plays an eminently important role. For the desorption of peptides, several very efficient matrixes have been found which produce a very fine crystalisation. There are now several responsive matrixes for DNA, however, the difference in sensitivity has not been reduced. The difference in sensitivity can be reduced by chemically modifying the DNA in such a manner that it becomes more similar to a peptide. Phosphorothioate nucleic acids in which the usual phosphates of the backbone are substituted with thiophosphates can be converted into a charge-neutral DNA using simple alkylation chemistry (Gut I G, Beck S. A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 1995 Apr. 25; 23(8):1367-73). The coupling of a charge tag to this modified DNA results in an increase in sensitivity to the same level as that found for peptides. A further advantage of charge tagging is the increased stability of the analysis against impurities which make the detection of unmodified substrates considerably more difficult. [0019] Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989. DESCRIPTION [0020] The invention provide a method for the analysis of biological samples for features associated with the development of lung cell proliferative disorders, characterised in that the nucleic acid of at least one member of the group comprising MDR1, APOC2, CACNA1G, EGR4, AR, RB1, GP1b beta, MYOD1, WT1, HLA-F, ELK1, APC, ARHI, BCL2, BRCA1, CALCA, CCND2, CDH1, CDKN1B, CDKN2a, CDKN2B, CD44, CSPG2, DAPK1, GGT1, GSTP1, HIC-1, LAP18, LKB1, LOC51147, MGMT, MLH1, MNCA9, MYC, N33, PAX6, PGR, PTEN, RARB, SFN, S100A2, TFF1, TGFBR2, TIMP3, VHL, CDKN1C, CAV1, CDH13, NDRG1, PTGS2, THBS1, TMEFF2, PLAU, DNMT1, ESR1, APAF1, HOXA5 and RASSF1 is/are contacted with a reagent or series of reagents capable of distinguishing between methylated and non methylated CpG dinucleotides within the genomic sequence of interest. [0021] The present invention makes available a method for ascertaining genetic and/or epigenetic parameters of genomic DNA. The method is for use in the improved diagnosis, treatment and monitoring of lung cell proliferative disorders, more specifically by enabling the improved identification of and differentiation between subclasses of said disorder and the genetic pre-disposition to said disorders. The invention presents improvements over the state of the art in that it enables a highly specific classification of lung carcinomas, thereby allowing for improved and informed treatment of patients. [0022] In a particularly preferred embodiment the present invention makes available methods and nucleic acids that allow the differentiation between squamous cell carcinoma, and adenocarcinoma and their respective adjacent lung tissues. Continue reading about Method and nucleic acids for the analysis of a lung cell proliferative disorder... 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